Control System -inkwell Coursework Example | Topics and Well Written Essays - 4250 words

Control System -inkwell - Coursework Example A review of this system is targeted at enhancing how the company handles its employees’ remuneration and other work packages. In this report, the chosen system for review is the payroll system, whose investigations are expected to provide a summation of the entire accounting system that is in operation in the organisation. A study of Inkwell’s payroll system shall entail an in-depth examination of the organisational activities such as the tax management, tracking of taxes that have been withheld, analysis of the record of hours employees work, and a record of the paychecks issued to employees (Bragg, 2006.p.125-129). To assess the payroll system being used by the company with the motive to provide recommendations for its improvement in efficient employee management. The payroll system, being a core accounting system is essential to the management as it streamlines the organisation’s ability to stay on top of its regulatory and legal responsibilities. Therefore, this report shall seek to identify the weaknesses within the organisation’s payroll system, and determine the needs that require improvement. Therefore, the report shall outline the cost-benefits analysis of implementing the payroll system’s improvements in Inkwell Limited (Bragg, 2003.p.85-92). It is expected that as a result of this review, it would be possible for the payroll system being used to effectively process payroll reports that can be used to make tax withholding summaries. The covering of this system’s evaluation in this report has been achieved as part of an accounting assessment, which is part of the AAT level 4 accounting. 2.1 Inkwell Limited is a Limited Company with a larger section of its operations majored in the private sector. However, its specialisation in the re-manufacturing or re-cycling of used laser toner and printer cartridges. The company’s sales targets are achieved by distributing its products through a nationwide chain that comprises over 60 high

Sociology - Values are the cultural conceptions Essay

Sociology - Values are the cultural conceptions - Essay Example Posted speed limits are an example of a formal norm where there is a written rule and a prescribed punishment for violation. Sanctions are the punishments and rewards that are received for violating or obeying society's norms. Sanctions will vary according to the value society places on a norm. The sanction for speeding will be more severe than the sanction for wearing a hat indoors. Since it is a norm for students to get good grades, if I work hard I will be rewarded with a good grade. Folkways are informal norms that govern the everyday actions of the members of a society. They exist to promote a unified acceptance of right and wrong and serve to regulate the behavior of society. When a man opens a door for me it is a folkway that has been used to reinforce the traditional male dominance in society. Mores are the norms that a society deems especially important. They encompass the most treasured values of a society. In America children are highly valued, as is their welfare. The laws against child abuse are social mores. Ascribed status is the significance that society places on a person's social standing without respect to their accomplishments or talents. Ascribed status may involve age, gender or race. In America, the white male has been given an ascribed status based on race and gender. Ascribed status is continually being challenged in society as illustrated by the feminist movement. AchievedAchieved Status Achieved status is attained through one's own actions and efforts. The status may be positive such as mayor or negative such as convicted felon. Ascribed status can influence our possibility of achieved status. George Bush has an ascribed status of being white, male, and from a prominent family. However, he did make the effort to become president and that is his achieved status. Master Status Throughout a person's life, they may gain several different stations in life. A successful corporate CEO may find himself in prison or as an ex-convict. The way that society views his status is the master status. Martin Luther King has the ascribed status of being black. He also had the achieved status of minister, civil rights leader, and convict. His status as a civil rights leader outweighs his status as a convict and becomes his master status. The master status may change with time and with the individual members of the society. Role Strain Role strain arises when a social position demands conflicting requirements. A business manager may have the task of hiring the best people available, yet is given a limited budget to work with. His role is to fill the vacancies with qualified people, and also as a budget watchdog. The President often comes under role strain while acting as Commander in Chief and as a representative of the people, each having conflicting desires. Role Conflict Role conflict arises when a person holds two or more social positions that have conflicting roles. By fulfilling the role of one position they are required to violate the role of another. In the workplace, people who are promoted are often faced with managing people that used to be their peers. They are placed in the position of fulfilling the role of supervisor or the role of trusted friend, which may result in a conflict. Works Consulted Henslin, James M. Essentials of Sociology: A Down to Earth Approach. 6th ed. Upper Saddle River, NJ: Allyn & Bacon,

Modernization Theory and Dependence Theory Analysis

Modernization Theory and Dependence Theory Analysis In this day and age the rapid development of the word and the growing assimilation of countries can hardly fail to affect the development of new theories which attempt to explain the relationship between countries and the existing inequality between developed countries and countries of the third world. Two theories which analyze the development in third world countries are the modernization theory and the dependence theory. These two theories, while being rather different, still have several similarities in their views on the modern world and relationships between developed and developing countries. As Alvin So explained, there are three chief and historical essentials which were constructive to the foundation of the modernization theory of development after the Second World War.First, the United States rose as a superpower.While other Western nations, such as Great Britain, France, and Germany, were undermined by World War II, the United States came out of the war stronger then before, and became a world leader with the execution of the Marshall Plan to reconstruct Western Europe.[2]Second, the idea of communist began to move throughout the world.What was once the Soviet Union spread its influence to Eastern Europe, China, and Korea.Third, there was the breakdown ofEuropean colonial empires in Asia, Africa and Latin America, creating numerous new nation-states in the Third World.These budding nation-states began searching for a form of development to support their economy and to improve their political independence. The modernization theorys intellectual lineage has been traced back to Aristotle. Aristotle first recommended that states, just as plants, went through a natural pattern of growth. Just like Aristotle, Americans in the early Republic assumed that if societies grow in a natural manner, they must also perish. The thought that the progression of human development could be understood and controlled dates to the early nineteenth century, when France and Britain were struggling to bring back their trade empires. Since then it has tended to reappear at times and places where systems of dominance required explanation and rationalization. The modernization theory looks at the internal factors of a country with the assumption that, with aid, â€Å"traditional† countries can be developed in the same way more developed countries have. The modernization theory tries to recognize the social variables which cause social growth and development of societies, and then tries to explain the social evolution. In order for a country to have a profitable, sophisticated, modern economy the country must follow a pattern of development. This is a very systematic theory as it means do one thing and another will happen. In order for this to happen, there need to be prerequisites for takeoff that will lead to takeoff in which will lead to mass-consumption(Mahler 45). A missing component of this theory is that the modernization theory assumes all countries will follow the set path to development. There are actually numerous variables in which will affect a states ability to in fact develop. An example of this is the fact that Mexic o is geographically designed in a way that will cause it to have a weak economy due to the deserts, forests, and mountains. This makes it so that only 12% of the land is arable. The fact that there are no major rivers doesnt help either. These issues all help to making it tricky for Mexico to develop because it restrains transportation, which in turn weakens the possibility of exporting and importing goods in a proficient manner.Another problem with the modernization theory is that it assumes that all states have the necessary preconditions to develop. This is not true as many states do not have proper leaders and government. The explanation for this is that if a state is controlled by weak leadership, it will in turn influence its ability to develop. For example, Saddam Hussein, made it so that his country could not develop because he took all of the wealth for himself. Perhaps, if Hussein had spread the wealth throughout his country, this will have helped education and increased i nvention. This could have made it so that his country developed in a more efficient manner. One policy implication the modernization theory suggests is that the third world countries should look up to the developed western nations, while the Western countries should pass on more modern values, institutions, technology, and financial investment to the Third World countries. Another implication is that in order for the third worlds to develop, they should be moving along the path that the United States has traveled, hence move away from the ideas of communism. (READING) A theory in which is opposed to the Modernization model which was created largely as a response to it is the Dependency theory. Dependency theories developed in opposition to the optimistic claims of modernizationtheory which saw the less developed countries being able to catch up with the West. They stressed that Western societies had an interest in maintaining their advantaged position in relation to the LDCs and had the financial and technical wherewithal to do so. A variety of different accounts of the relationship between the advanced and less developed states evolved within the broad framework of dependency theory, ranging from the stagnationism and ‘surplus drain theory of Andre Gunder Frank (which predicted erroneously that the Third World would be unable to achieve significant levels ofindustrialization), to the more cautious pessimism of those who envisaged a measure of growth based on ‘associated dependent relations with the West. The major contribution to dependency theory was undoubtedly that of Frank, a German economist of development who devised and popularized the phrase ‘the development of underdevelopment, describing what he saw as the deformed and dependent economies of the peripheral states-in his terminology the ‘satellites of the more advanced ‘metropolises. InCapitalism and Underdevelopment in Latin America(1969), he argued that the Third World was doomed to stagnation because the surplus it produced was appropriated by the advanced capitalist countries, through agencies such as transnational corporations. Frank himself insisted that growth could only be achieved by severing ties with capitalism and pursuing autocentric socialist development strategies. According to the dependency theory, the Global North exploits the Global South. One reason for this is that the south is highly dependent on the wealth of the north; therefore unable to advance themselves because of the vicious cycle that then ensues. An example of this vicious cycle can begin with a country being very poor and/or economically unstable. They then allow a multinational corporation to set up camp in one of their cities. This leads to many new jobs for this city, but the people are hired for very poor wages. Then the products that are produced get siphoned off by the Global North, in turn preventing that states â€Å"mass-consumption† abilities which is a generalized way that the south gets exploited by the north and the multinational corporation comes out making huge profits at the expense of desperate people just trying to survive and willing to work for pennies. The depencde theory has several implications. First, Promotion of domestic industry and manufactured goods. By imposing subsidies to protect domestic industries, poor countries can be enabled to sell their own products rather than simply exporting raw materials. Second, Import limitations. By limiting the importation ofluxury goodsandmanufactured goodsthat can be produced within the country, the country can reduce its loss of capital and resources. Thrid, Forbidding foreign investment. Some governments took steps to keep foreign companies and individuals from owning or operating property that draws on the resources of the country. In conclusion, both theories admit the leadership of western countries and their currently dominant position in the modern world, while undeveloped countries are characterized by socio-economic and political backwardness. At the same time, the two theories agree that the cooperation between western countries and developing countries is constantly growing and leads to their integration. However, it is necessary to underline that Modernization theory views such cooperation and integration as a conscious and voluntary act from the part of developing countries, for which modernization in the western style is the only way to overcome the existing backwardness, while supporters of Dependency theory argue that such cooperation and integration is imposed to developing countries by more advanced western countries, which simply attempt to benefit from their cooperation with developing countries and their westernization becomes a way of the establishment of control over and growing dependence o f developing countries on developed ones. Regardless, the existing differences, both theories still raise a very important problem of relationships between developed and developing countries and the dominance of western countries and western civilization in the modern world.

Narcissism in John Milton’s Paradise Lost Essay example -- Milton Para

Narcissism in John Milton’s Paradise Lost When Eve eats the forbidden fruit of the Tree of Knowledge, her decision to tell Adam of her disobedience turns on two suppositions. If her transgression is kept secret from God, Eve's augmented knowledge might increase Adam's love for her, and perhaps cause her to be more equal or even superior to Adam. Even though Eve was created comparable to Adam as his helper, she refers to Adam as her "Author and Disposer." Furthermore, she says that while God is Adam's law, Adam is her law. Apparently, Eve chafes under this arrangement, as she wraps up her evaluation of not telling Adam of her sin with, "for inferior who is free?" However, her death is assured if God has seen her wrongdoing. In this alternative, God may provide Adam with another woman, rendering Eve extinct. Eve finds unendurable the possibility that Adam will father children with a new Eve. Eve's consideration of either alternative depends on her narcissism and her need to be loved, even worshiped. Milton's Eve, like Narcissu s, is infatuated with herself. Created in Adam's image, Eve draws Adam's love, his narcissism projected onto Eve. Inexperienced with women's wiles, uxorious Adam falls. Having created Adam in his own image, the Lord God commanded Adam not to eat of the Tree of Knowledge of good and evil. After the Lord God created Eve from Adam's rib, Milton's Adam warns Eve that the consequence of eating the tree's forbidden fruit will be the knowledge of death. From the Bible and Milton's text, it is apparent that Eve hears directly only from Adam about the forbidden fruit. It is significant that God sends Raphael to "converse with Adam," to warn him of the fall of Satan and his companions, and to alert Adam to the ... ...rcissism engenders the desire to be worshipped as a Goddess. Wanting deity for both, Eve chooses to induce Adam to eat because she is resolved that Adam shall share her fate. Eve's female charms seduce Adam. He desires Eve more than he loves God, and he eats freely of the fruit. Adam and Eve's ardour, once based on mutual respect, turns to carnal lust. God's Son berates Adam for subjecting himself to Eve's will. Why did Adam obey Eve, who is in no way superior to Adam in reason and other faculties of the mind? Adam has sinned against God; Eve has sinned against God and Adam. Only when Mary of the seed of Adam and Eve conceives the Son of God does God extend his grace to mankind, permitting narcissistic Eve and uxorious Adam's progeny to enter heaven. Works Cited and Consulted Milton, John. Paradise Lost. Ed. Merritt Y. Hughes. New York: Odyssey Press, 1962.

Inoculation of an Egg

1. EGG INOCULATION The fertile hen’s egg can be used to cultivate and propagate various types of viruses. Because of the ability to alter their tropism and to adapt to a new host species, many viruses become capable of growing in chick embryo tissues wherein they frequently attain a much higher concentration than in the tissues of the natural host. STRUCTURE OF AN EGG The extra-embryonic membranes of the chick embryo arise from three germinal layers: the endoderm, mesoderm and ectoderm (Fig. 1).The dorsal somatopleure consists of ectoderm on one side and mesoderm on the other side while the splanchnopleure consists of mesoderm and endoderm. By a process of folding, the somatopleure gives rise to the chorion and amnion while the allantois and yolk sac membranes develop from the splanchnopleure. The amnion arises from the head and caudal regions of the embryo, the membrane being reflected back to form the chorion. the amniotic membranes grow rapidly and fuse to form the amniotic sac by the 5th day. The allantois grows out as a bud from the hind gut of the embryo and enlarges rapidly.By the 10th day the allantois becomes attached to the outer layer of the amniotic sac and the inner layer of the chorion to form the chorioallantoic sac (CAS) which separates the chorion from the amnion. The fused chorionic and allantoic membranes are referred to as the chorioallantoic membrane (CAM). Because the CAS represents a diverticulum of the gut, it serves as the excretory receptacle for the embryo. It contains from 5 to 10 ml of fluid with dissolved solids, the solution being clear in early stages but becoming turbid after the 12th day due to the presence of urates.The CAM is the respiratory organ of the embryo and thus is richly supplied with blood vessels. The embryo is surrounded by the amniotic sac and lies bathed in about 1 ml of amniotic fluid. The amniotic fluid, which contains much of the albumin in the egg, serves as a source of protein which is ingested durin g swallowing movements the embryo is seen to make from the 9th day onward. The air-sac is present in the blunt end of the egg. Underlining the shell is the fibrous egg shell membrane. In the beginning stages of development, the chick embryo can be recognized with difficulty as a small dark area attached to the yolk sac.After 4-5 days the embryo can be readily detected by candling. After the 10th day, the embryo development, rapidly increase in size and feathers appear. The respiratory tract develops between the 12th and 15th days. If the egg remains uninoculated and is maintained in a humid 38oC environment, it will hatch on the 21st day of life. Inoculation Procedures The methods described below for the inoculation of the chick embryo do not comprise a complete list but represent those that are practiced most commonly. Likewise, while there are a number of techniques for inoculation by each of the routes listed, only the one most widely used is described.A. Yolk Sac Chlamydia and r ickettsia grow readily in the yolk sac (YS) membranes. Although some of the smaller viruses are inoculated by the YS route, they invade and replicate in the tissues of the embryo itself rather than in the YS tissues. a. Candling and drilling. Fertile eggs that have been incubated for 5 to 7 days are suitable since the YS is relatively large at this time. The eggs are candled and the boundary of the air sac penciled in. The shell over the air space, which is referred to as the shell cap, is disinfected by an application of iodine to one small area.When the iodine is dried, a hole is made through the shell over the center of the natural air space by means of a drill or egg punch. b. Inoculation and incubation. By means of a syringe fitted with a one and one-half to two inch 23 gauge needle, the inoculum is deposited in the YS by passing the needle through the hole in the shell cap and directing it downward to its full length parallel to the long axis of the egg. From 0. 2cc to 0. 5cc is usually inoculated. the hole in the shell is then sealed with tape and the eggs are incubated at 37oC. c. Harvesting Procedure.The egg is placed in a container which maintains it in the upright position during the harvesting procedure. The shell is cracked with sterile forceps and the cap lifted off. The exposed membranes are torn away. If the YS membranes are to be harvested. The contents of the egg are quickly emptied into a sterile petri dish. The YS is usually ruptured in the process. The YS membranes, which are easily recognized by their deep yellow color, are detached from the embryo and separated from the chorioallantois with sterile forceps and quickly transferred to a sterile petri dish.When the embryo is to be harvested, it is withdrawn by hooking the curved end of a dental probe around the neck. It is then separated from the adherent membranes with sterile scissors and transferred to a sterile petri dish. B. Chorioallantoic Sac (CAS) The influenza and the newcastle dis ease viruses and most other viral agents which cause respiratory infections grow readily in the endodermal cells of the allantoic sac wall and are liberated into the allantoic fluid. The encephalomyelitis viruses and the mumps virus also multiply readily when inoculated by this route. . Candling and drillings. Embryonating eggs which have received a preliminary incubation from 9 to 11 days are candled and the boundary of the air space penciled in. The eggs are held in the upright position with the air sac uppermost. A point is selected a few millimeters above the floor of the air space on the side of the egg where the chorioallantois is well-developed but free of large vessels. Iodine is applied to the area around the site. A hole is then drilled or punched through the shell. b. Inoculation and incubation.A one-half inch 26 gauge needle, fitted to a small syringe containing the inoculum, is inserted into the allantoic cavity by passing it through the hole in the shell parallel to th e long axis of the egg or at an angle directed towards the apical extremity. From 0. 1cc to 0. 2cc of inoculum is injected into each egg. The hole in the shell is then sealed with tape and the eggs are incubated. c. Harvesting of allantoic fluid (AF). In order to avoid hemorrhage into the AF while harvesting, the eggs are chilled in the refrigerator from 4 to 6 hours prior to the harvesting procedure.While harvesting, eggs are held in an upright position and the shell over the air sac is removed with sterile forceps. The floor of the air space is exposed. With a pair of small sterile curved forceps these membranes are torn away. In order to facilitate the harvesting of the AF the embryo is displaced to one side by placing the forceps against the embryo with the tips toward the shell wall. The AF can then be readily aspirated with a 5 ml or 10 ml sterile pipette. C. The Chorioallantoic Membrane (CAM) Nine to 12 days old embryonating eggs are candled and an area over the most vascular portion on the side of the egg marked with a pencil.The shell is disinfected with iodine over this point and also the air sac end. A hole is carefully punched over both these locations. The hole on the side of the egg must penetrate both the shell and the inner shell membrane. A small amount of fluid may exude from the hole if the inner shell membrane is penetrated. While candling the egg with its long axis in the horizontal position, a piece of rubber tubing is placed firmly over the hole in the end of the egg. Suction is applied until the air sac collapses in the end but reappears on the side of the egg.When this false air sac is confirmed by candling, the CAM is ready for inoculation. The CAM and inner shell membrane are usually tightly adherent by 9 days of incubation, and the inner shell membrane may consequently by dropped as well as the CAM. This is unacceptable since the inoculum will fall on the inner shell membrane and not the CAM. To avoid this, a drop of sterile PBS is placed over the newly punched hole in side of egg to soften the membranes. An alternate method is to drop the CAM at 7-8 days of incubation, then wait until 11-12 days before inoculation. a. Inoculation and incubation.By carefully passing the needle through the inner shell membrane from 0. 1cc to 0. 2cc of inoculum is dropped on the chorioallantois with a 1cc syringe fitted with a 22 or 23 gauge one-half inch needle. In very critical studies the egg should be candled during this procedure to insure that the inoculum is deposited on, rather than through, the membrane. After inoculation the egg is gently rocked in order to spread the inoculum uniformly over the surface of the CAM. The opening in the shell is covered with a small square of scotch tape and the inoculated eggs are incubated in a horizontal position with the hole uppermost. . Harvesting of the membrane tissues. The egg is placed in the horizontal position with the hole uppermost. Iodine is applied to the area around the w indow with a cotton swab and the tape then peeled off. The surrounding shell is broken away with sterile forceps and the chorioallantois exposed. The membrane is grasped with forceps, detached with scissors and quickly transferred to a sterile Petri dish. D. Amniotic Sac This method is used principally for the isolation of the influenza virus from throat washings. The embryo during the course of its development wallows the amniotic fluid, thereby bringing the inoculated virus which it contains into contact with the tissues of the respiratory and intestinal tracts where multiplication presumably occurs. After incubation amniotic fluid is then â€Å"subpassaged† by the CAS route (Fig. 2). The amniotic route of inoculation is used also for the isolation of the encephalomyelitis virus. a. Candling and drilling. Embryos from 13 to 15 days of age are used. The position of the embryo is determined by candling and a point on the shell over the air space on the side of the egg on whic h the embryo is situated is marked.The site is prepared in the usual manner and a hole is drilled or punched as for yolk sac inoculation. b. Inoculation and incubation. A 1cc syringe fitted with 1 3/4 inch 24 gauge needle is used for the inoculation. The egg is placed horizontally on the candler, the needle is introduced and gently stabbed in the direction of the embryo. Penetration of the amniotic sac is indicated by a sudden movement of the embryo. The needle is then withdrawn slightly and from 0. 1cc to 0. 2cc of the inoculum injected. the hole in the shell is sealed with tape and the eggs are incubated in the vertical position. . Collection of amniotic fluid. The shell is removed as for the allantoic and yolk sac routes of inoculation. A few drops of saline are placed on the floor of the air space to render the membrane transparent. Using the eyes of the embryo as a reference point, the amniotic fluid is aspirated by means of a Syringe fitted with a short 23 gauge needle. E. Mis cellaneous Routes ofInoculation a. Intravenous. This method is not used commonly, although it is the method of choice for the isolation of bluetongue virus. A large vein is located and marked in 12-14 day embryos.A rectangular piece of shell directly over the vein is removed and a droplet of sterile mineral oil is placed on the inner shell membrane so as to render it transparent. A 27-30 gauge five-eight inch needle fitted to a small syringe is introduced through the membrane into the vein in the direction of blood flow. From 0. 1 to 0. 5cc of inoculum is then injected. Incubation and harvesting of the embryo is carried out as already described. b. Intracerebral. This route may be used in the studies of pathologic alterations of the brain following infection. Eight to 14 day embryos are usually used.The viruses of herpes simplex and rabies may be cultivated by this method. Egg Inoculations. Materials needed: Embryonated eggs, 11-12 days A paramyxovirus, PI3 or Sendia virus Vaccinia virus Crystal violet Appropriate syringes and needles Egg candlers, egg punches Iodine disinfectant and swabs, cellophane tape Instruments, petri dishes Procedure: 1. Inoculation of dye into CAS a. Candle 11-12 day embryonated egg, mark boundaries of air sac with pencil. Just above air sac, choose a point devoid of vessels and mark with a pencil. bDisinfect egg shell at this point with iodine.Let dry before next procedure. c. Drill a small hole with an egg punch at the appropriate marked point (be careful-don’t break the shell). d. Inject 0. 1 – 0. 2ml dye into the CAS as described and illustrated. e. Place a small piece of cellophane tape over the hole. The egg would be ready to incubate if the inoculum had been virus. f. Candle the dye-inoculated egg to establish that the inoculum is in the correct place. Watch the inoculum spread through-out the confines of the CAS. g. Break the egg and pour the contents into a petri dish. Observe where the dye is.Identify the CAM, YS, amnion and embryo. If inoculated properly only the CAS should contain dye. 2. CAS inoculation of virus a. Follow procedures for CAS inoculation of a dye, except the inoculation should be 0. 1 ml of a live paramyxovirus. Place tape over the inoculation hole and incubate. b. Candle egg daily to determine embryo viability. If the embryo dies within 2 days of inoculation, it usually indicates bacterial contamina-tion or trauma. c. If the embryo dies after 2 days, refrigerate as soon as death is noted until the next laboratory period. 3. CAM inoculation of vaccine virusWarning;If you have not had a successful smallpox vaccination, have eczema or evidence of immune deficiency, contact the instructor before handling this virus. Be careful! Do not get this virus in your eyes! Vaccinia virus is the live virus vaccine for smallpox. While less pathogenic than smallpox or variola virus it can still cause serious or uncomfortable lesions if mishandled. a. Two 11-12 days embryonated eggs will be supplied to each group of students. Drop the membrane on both of the eggs according to the instructions and illustrations. b. Inoculate 0. ml vaccinia virus onto the dropped CAM. be sure to go through the inner shell membrane but not through the CAM. Rock the egg to distribute the inoculum over the entire floor of the false air sac. Cover the hole with tape and incubate in a horizontal position with the hole uppermost. 4. Harvesting of embryonated eggs (next laboratory). a. Follow instructions for removal of CAS fluid. Try to keep blood vessels from rupturing. Remove CAS fluid aseptically in a sterile pipette. Expel fluid into a sterile vial. This fluid will be used for the hemaggulatination exercise later.It can be frozen if necessary. Use the last drop of CAS fluid to inoculate bacteriological media to check for contamination. b. Harvest CAM as per instructions. Place the membrane in a petri dish and lightly pour PBS over the membrane until it flattens out and the pocks are cl early visible. c. Important. All fluids, instruments, and other things that have come into contact with virus-infected tissues must be properly sterilized. Follow carefully the instructor’s remarks for proper disposal of all materials. Be sure to disinfect your workspace with disinfectant when cleaning up. 1. INFECTIVITY ASSAYS The concentration of a suspension of virus is usually determined by measuring its infectivity. There are two types of infectivity titrations: the quantal assay, which depends upon an all-or-none does response, and the quantitative assay, which utilizes a plaque, pock or lesion count in which the effect of a single infectious virus particle is seen as a visible localized change in a background of normal cells. A. Quantitative Assay2 This method determines the actual number of infectious units (virus particles) in a given suspension.This type of enumerative response is assessed from focal lesions such as plaques in cell cultures, pocks on the CAM of chic k embryos or local necrotic lesions on a plant leaf. The number of infectious units per unit volume can be calculated, and this is referred to as the titer. With plaque assays, the titer of the original virus suspension is stated in terms of the number of plaque forming units (PFU) per ml. Ex. Fifty plaques on a 10-5 dilution of original suspension were counted. A 0. 1ml inoculum was used. #PFU/ml. of original volume No. of plaques = ——————————— dilution) x (Vol. of inoculum) =5. 0 x 107 PFU/ml=50 x 106 = or 105 x 0. 1 B. Quantal Assay This assay estimates the concentration of infectious particles by allowing them to replicate in a suitable host so that one infectious unit can be detected by the amplification effect of the infection. The actual number of infectious particles introduced into the test unit is unknown and may vary even between duplicates of the same dilution. To determine quantal infectivity t iters, mutiple replicate tests are used for each dilution of original suspension until the infectivity is diluted out.The result gives the dose necessary to produce a defined response. This response is usually based on a 50% end point, which is the dilution at which 50% of the test animals, eggs, or cell cultures react to the virus. Computation of the 50% end point is based on the presence or absence of a predetermined criterion, i. e. death (Median Lethal Dose or LD50), infectivity (Median Tissue Culture Infective Dose or TCID50, Median Egg Infective Dose or EID50), etc. The criterion must be either present of absent: either the animal is dead or alive, or the cell culture is infected or not infected.There are no plus/minus or graded reactions. This method does not measure the exact number of virus particle but only whether or not virus is present at a particular dilution. There are two formulas that can be used to determine 50% endpoints; the Reed-Muench and the Spearman-Karber me thods. Both are demonstrated here using the same data. 1. Reed-Muench Method Accumulated Values |Virus Dilution |Morality Ratio | | | | | | | |(a) |(b) |Died |Survived Died |Survived |Ratio |Percent | | | |(c) |(d) |(e) |(f) |(g) |(h) | |10-1 |6/6 |6 |0 |17 |0 |17/17 |100 | |10-2 |6/6 |6 |0 |11 |0 |11/11 |100 | |10-3 |4/6 |4 |2 |5 |2 |5/7 |71 | |10-4 |1/6 |1 |5 |1 |7 |1/8 |13 | |10-5 |0/6 |0 |6 |0 |13 |1/13 |1 | | | | | | | | | | At the 10-3 dilution, 5/7 or 71% of the accumulated test animals died, and at the 10-4 dilution, 1/8 or 13% died (columns g and h). The 50% endpoint, therefore, lies somewhere between the 10-3 and 10-4 dilutions. The final calculation requires interpolation between these two values. The formula for doing this is: (% mortality at dilution next above 50%) – (50%) ————————————————————— = proportionate distanc e (% mortality at dilution next above 50% – Mortality at at dilution next below or 71-50 21 —- = — = 0. 36 proportionate distance 71-13 58The dilution factor must also be considered, i. e. , 2-fold, 4-fold, 10-fold, etc. and the proportionate distance corrected (multiplied) by the log10 of the dilution factor (2-fold = 0. 3, 5-fold = 0. 7, 10-fold = 1, etc. ) The final estimate is determined by this formula: Negative log10 of LD50 end point = negative log of dilution above 50% mortality plus the proportionate distance factor (corrected for dilution series used) Negative log of dilution above 50% mortality –3. 00 Proportionate distance (0. 36) x dilution factor (log10-1= -1)= – 0. 36 Negative log LD50= -3. 36 LD50=10-3. 36 antilog of 10. 36=2. 29 LD50 titer -3. 36 =2. 29 x 103 / volume inoculated LD50 Calculation Inoculum: |DILUTION |DEAD |ALIVE |CUMULATIVE DEAD |CUUMULATIVE ALIVE |LD50= | |EXAMPLE |10-1 |6 |0 |17 |0 |(a-b)(c+d) | | | | | | | |2[(ax d)-(bxc)] | |Test System: |10-2 |6 |0 |11 |0 |=(3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | |Date Inoculated |10-3 |4 |2 |5 |2 |=24 =0. 36 | | | | | | | |66 | | |10-4 |1 |5 |1 |7 |LD50 = 3. 36 | | |10-5 |0 |6 |0 |13 | | |Inoculum |Dilution |Dead |Alive Cumulative Dead ( |Cumulative Alive ( | | | | | | | | |LD50= | |Test Systems: | | | | | |(a-b) (c+d) | | | | | | | |2((a x d) – (b x c)( | | | | | | | | | |Date Inoculated: | | | | | |= (3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | | | | | | | |=___________ | | | | | | | | | Inoculum |Dilution |Dead |Alive |Cumulative Dead ( |Cumulative Alive ( | | | | | | | | |LD50= | |Test Systems: | | | | | |(a-b) (c+d) | | | | | | | |2((a x d) – (b x c)( | | | | | | | | | |Date Inoculated: | | | | | |= (3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | | | | | | | |=___________ | | | | | | | | | 2. Spreaman-Karber Method Estimation of the 50% endpoint by the Spearman-Karber method is much simpler. The formula is: Negative log10 of LD50 = X – d (P-0. 5) here X = log10 of the highest concentration used (lowest dilution); d = log10 of the dilution factor, and p = sum of % mortality at each dilution100 Using the same data chart above the following number are obtained: d(10-1)(do-2)(10-3)(10-4) Neg log : LD50 = 1. 0 -1(100 + 100 + 66 + 17)-0. 5 100 = -1 [1(2. 84-0. 5)] = -1 2. 34 = -3. 34 LD50 antilog of 10. 34 = 2. 19 LD50 titer= 2. 19 x 103 volume inoculated Note that the two methods produce slightly different results using the same data. The Spearman-Karber method is considered to be the more accurate. The Spearman-Karber method can be simplified even more if signs are neglected and common sense used.This formula is: Neg log LD50 = X + d (P + 0. 5) Where X = log10 of highest dilution showing 100% mortality; d = log10 of dilution factor; p = proportion of positives above dilution X Xdp Neg. log LD50 = 2 + 1 (4/6) = 1/6 + 0. 5) = 2 + 1 (. 67 + . 17 + 0. 5) = 2 + 1. 34 = 3. 34 The appropriate sign can then be inserted: LD50 = 10-3. 34 These formulas can also be used to estimate 50% endpoints in neutralization tests. Here, absence of the predetermined criterion is counted and used for the calculations. III. SEROLOGIC TECHNIQUES A. Hemagglutination Many viruses or viral antigens are capable of specifically and non-covalently binding to receptors on the surface of red blood cells (RBCs).When the right volumes of these viruses and RBC’s are mixed, the viruses bridge the RBCs to form a lattice which settles out of suspension in a uniformly thin shield on the bottom of a test tube or conical well. This phenomenon was first described by Hirst in 1941 and is known as hemagglutination (HA). the HA titer of a virus can be determined by mixing serial dilutions of a virus with a constant amount of RBCs which are usually prepared as a 0. 25%-1. 0% suspension in physiological saline. the highest dilution which agglutinates the RBCs is the endpoint. The HA titer is the reciprocal of the endpo int dilution, and that dilution is said to contain one HA Unit (HAU) of virus in the original volume.Unagglutinated RBCs sediment to a packed disc (â€Å"button†) on the bottom of the test tube or well. The viruses known to cause hemagglutination are heterogeneous but can be grouped according to the nature of their hemagglutinating protein (hemagglutinin). The hemagglutinin on the virion of influenza and the paramyxoviruses is a glycoprotein. These viruses, but not any others, also carry an enzyme, neuraminidase, which destroys the glycolipid receptors on the RBC surface and allows the virus to elute (unless the HA is carried out at a temperature too low for the enzyme to act). Certain toga and Coxsackie viruses possess a hemagglutinin but do not possess a neuraminidase-like enzyme. astidious conditions are necessary for these viruses to hemagglutinate and usually cells from only a very few species can be can be used. Vaccinia virus has a lipoprotein hemagglutinin associated with a soluble fraction separable from the viral particle itself. Some viruses agglutinate RBCs from a limited number of course and some HA reactions require careful control of pH, temperature and ionic conditions (see Table 5-1, p. 100-101, Rovozzo & Burke). We will perform the HA test with a paramyxovirus which will agglutinate human type O, bovine, guinea pig or chicken RBCs over fairly broad ranges of pH (6. 0-8. 0) and temperature (4-25oC). Material needed: 1 cc syringes 0. 025 ml microtiter tips 0. 25 ml microdiluters Microtiter plates with 96-V-bottom wells Phosphate buffered saline (PBS) Paramyxovirus, in form of allantoic fluid harvested 48-96 hr after infection of 10-day embryonating eggs Washed RBCs, 0. 5% in PBS Go-no-go test papers Dilution tubes and 1 ml pipets *Use only the top half the microtiter plate. Procedure: 1. With the micropipet (a microtiter tip attached to a 1cc syringe) held vertically, dispense 0. 025 ml (1 drop) PBS each into columns 2 through 12 of rows A. B, C, and D of the microtiter plate. Also put 0. 025 ml PBS into wells 1 through 4 of row H for controls. 2. Make a 1:10 dilution of virus in PBS in a dilution tube.With the same pipet used to dispense PBS, put 0. 025 ml of the 1:10 virus dilution into each well of columns 1 and 2, rows A-D. 3. Test the delivery volume of the microdiluter by immersing the tip in PBS then touching to the center of a circle on the go-no-go paper. Now begin dilutions by immersing the dry diluter in well two, rotating to mix and pick up 0. 025 ml of fluid, and transferring to well three. Continue rotating and transferring through row 12. Two lines may be diluted simultaneously if desired. After removal of 0. 025 ml from row 12, dip the diluter in disinfectant, then distilled water, then flame. Do not flame with protein or salt in the diluter.Do not add virus to the controls. 4. With a new syringe and tip add 0. 025 ml (1 drop) 0. 5% RBCs (mix suspension well before pipetting) to every well, includin g controls. Mix well by running a hard object down the underside of the plate. 5. Allow to stand at room temperature until the controls and higher virus dilutions have RBCs settled into a â€Å"button† in the point of the V, and positive wells have RBCs uniformly spread over the entire bottom of the well. This will take 1-2 hr. Then refrigerate the plate. 6. Read the HA titer as the reciprocal of the dilution of the last well showing positive HA. Calculate the dilution which contains 4HAU/0. 25ml for use in the HI test.Be sure to check controls for spontaneous agglutination. B. Hemagglutination Inhibition Viral hemagglutination may be inhibited in several ways. By combining with viral antigens which normally interact with RBC receptors, specific anti-viral antibodies can prevent the virus cell interaction which normally brings about hemagglutination. Since infection with a virus will elicit production by the host animal of antibodies directed against each virus-induced protei n, including the hemagglutinin, inhibition of hemagglutination by an animal’s serum indicates that the animal has been infected by the virus. A high HI titer may indicate that the infection was recent.A four-fold rise in titer between two serum samples taken a few weeks apart (as during acute and convalescent phases of a disease) indicates that infection occurred during the period between the sampling times. If the viral hemagglutinin is also the protein by which the virus attaches to cells susceptible to infection, a high HI titer shows an animal to be immune to reinfection. HI is carried out in much the same way has HA. The serum is diluted in microtiter plates and each dilution is allowed to react with a constant dose of virus (usually four HAU) for an interval of 15 min to one hr before RBCs are added. The reciprocal of the highest serum dilution which inhibits HA is the HI titer.Several controls are necessary (1) The lowest dilution of serum used in the test must be incu bated alone with RBCs to determine if it contains heterophile antibodies which cause RVC agglutination. (2) The virus must be back titrated to see that the proper dose was added to the test wells. (3) A known non-immune serum from the same animal species must be titrated (usually before the HI test is performed) to see if it contains non-specific inhibitors. Heterophile antigens are a group of shared antigens with over-lapping specificities. They are found in some plants (corn, spinach) some microorganisms (Pneumococcus, E. coli), and some fish and animal tissues (carp, toad, guinea pig, horse, man etc. Heterophile antibodies against these antigens will cross react with cells and fluids from the above-listed species. In the case of RBCs as the heterophile antigen, if heterophile antibodies against them are present, hemagglutination will occur, possibly masking the presence of the hemagglutination-inhibition reaction caused by anti viral antibodies. Nonspecific inhibitors of hemagglu -tination may also be found in the serum of man and animals. Their nature differs for different viruses and even for different strains of viruses, such as influenza virus. Serum inhibitors also differ in different species. Inhibitors may be of low titer, or in some cases higher than the actual antibody titers, thus masking its diagnostic importance.Methods which have been used to remove inhibitors include: (1) Heating at 56-1/4 for 30 min, (2) treatment with receptor-destroying enzyme (neuraminidase), trypsin and/or periodate, (3) absorption with kaolin, (4) extraction with acetone, (5) precipitation of beta-lipoproteins with heparin and manganous chloride or with dextran sulphate and calcium chloride. No single method is universally applicable. Sometimes more than one method must be used. Antibody titers can be depressed by some of these procedures. The final control used is the RVC saline control to check for self-agglutinating RBC. Table 1. Example of HA and HI |Virus |Antigen So urce |RBC |Temperature |Non. Sp. Inhib.Removal | |Influenza A and B |CAS fluid or cell culture|Chicken, Human O |Room |Neuraminidase | | |fluid | | | | |Mumps |CAS fluid |Chicken |Room |Neuraminidase | |Coxsackie |Cells culture fluid |Fowl |Room |Kaolin | |Rubella |Cell culture fluid |one-day old chicken, goose |4oC |Heparin and Manganous chloride| |Adenoviruses |Cell culture fluid |Rat, rhesus monkey |37oC/room |Not required | Procedure 1.Add one drop (0. 025 ml) PBS diluent to all wells of the microtiter plate. 2. You will be given three serums. One will be untreated, one treated for removal of non-specific inhibitors and/or heterophile antibodies, and one known negative serum. Your results will be compiled with the class results to clarify the total experiment. using the microdiluters, add 0. 025 ml test serum to well A of row 1 and 2. Dilute out to well H. The first well is a 1/2 dilution of serum and row H is 1/256. Add 0. 025 ml of the same test serum to row 7, the serum contr ol well. Dilute to well H as before. Carefully rinse the diluters and repeat with test serum 2 and test serum 3, sing rows 3, 4, 8 and 5, 6, 9 respectively. 3. Add 0. 025 ml of the challenge virus with the microdiluters to well A of rows 10 and 11. Dilute to well H. This is the antigen (virus) back titration and control. The highest dilution with complete hemagglutination is 1 HA unit. 4. Using the same â€Å"micro-pipet† as in #1, add another drop of PBS to all wells of rows 7-12. Empty the pipet and refill with the hemagglutinin (virus). Add one drop hemagglutinin to all wells of rows 1-6. Mix well. 5. Incubate at room temperature 30-60 minutes. 6. Using a new pipet, add one drop 0. 5% bovine RBC to all wells. Mix well. Store at 4oC and read the next day.Antibody titers are the highest dilution that inhibits hemagglutination (forms a distinct button). C. Hemadsorption Certain enveloped hemagglutinating viruses cause the insertion of viral hemagglutinins into the plasma memb rane of cells in which they are replicating. These modified areas of the cell surface are the sites at which progeny virus particles will mature. If agglutinable RBCs are brought into contact with hemagglutinin-containing surfaces of cultured cells, the RBCs will specifically bind to the infected cells. This phenomenon, known as hemadsorption, is particularly useful in detecting infection by viruses which cause little morphological change in infected cells. Procedure 1.Pour off medium from a tube of cultured cells infected with an orthomyxovirus or a paramyxovirus and from a tube of uninfected cells. 2. Wash monolayer thoroughly but gently with two rinses of 3 ml of physiological saline. 3. Add 0. 2 to 0. 5 ml 0. 5% bovine RBCs in saline. Allow RBC suspension to cover cell layer. Incubate at room temperature for 10-15 minutes. 4. Pour off RVC suspension and wash 2x with 2-3 ml saline. 5. Examine under microscope. Infected cells should have entire surface covered with RBCs. Non speci fic binding will cover only a few sites per cultured cell. SERUM NEUTRALIZATION The neutralization test estimates the capacity of a specific serum antibody to neutralize a virus biological activity.Major uses for this test include the identification of unknown virus or antibody, the determination of antibody levels, the comparison of antigenically related viruses and the study of the kinetics of antigen-antibody reactions. Viruses and the study of the kinetics of antigen-antibody reactions. Neutralization can occur by several mechanisms. Virus adsorption to cells may be inhibited by alteration of the configuration of cell receptor sites or by prevention of viral attachment. Virus degradation may be enhanced by interference with post-engulfment stages of virus replication, by prevention of release of functional virus cores into the cytoplasm or by the degradation of virus-Ab complexes within phagosomes.Also, complement-mediated reactions may enhance neutralization by production of le sions in the viral envelope. Several factors must be considered when performing a neutralization assay. Sensitivity of the test is related to the degree of susceptibility of the indicator host system to infection with the virus. The neutralization reaction is readily reversible by dilution with saline, by ultrasonic treatment or by lowering pH. Finally, the time required to reach equilibrium may vary with different systems. When performing the neutralization test two systems are used. The reaction system is incubation of virus and specific antisera until equilibrium is reached. The indicator system is the inoculation of the virus-Ab mixture into a susceptible host.If neutralizing anti-bodies are not present lesions such as pocks or plaques will be seen in the host. If neutralizing antibodies are present there will be no lesions. There are two techniques commonly used for the neutralization test. In the alpha procedure a constant serum concentration is added to serial log dilutions o f virus. The mixture is incubated and inoculated into an appropriate host system. In the beta procedure a constant virus concentration is incubated in serial two-fold dilutions of serum before inoculation into the host. The beta procedure is most commonly used because of its sensitivity, ability to measure antibody titer and its economical use of serum.The alpha procedure is not as sensitive and may be more subject to non-specific inhibition. It is more frequently used for comparative studies. Alpha Neutralization test Materials Needed: Flat-bottomed MT plate with bovine cell monolayer MT transfer plate with lid and holder MT tips 1 cc syringes serum samples stock virus MEM diluent dilution tubes sterile distilled water in beaker Procedure: Use aseptic technique. a. Make serial 10-fold dilutions of stock virus to 10-8 using MEM and dilution tubes (0. 2 plus 1. 8 ml). b. Using sterile 1 cc syringe and microtip add 1 drop (0. 025 ml) diluent (MEM) to rows 7 and 8 wells A-H, and rows 9 , 10 and 11 wells A and B of the transfer plate. c. Using the same syringe and microtip add 0. 25 ml of the virus dilutions to rows 1-8 as follows: 10-8 into wells H, 10-7 into wells G, and so on finishing with 10-1 in wells A. d. Using a new syringe and microtip add 0. 025 ml test serum A to rows 1 and 2 wells A-H, and row 9 wells A and B. Rinse syringe and microtip with sterile water and add 0. 025 ml serum B to rows 3 and 4 wells A-H, and row 10 wells A and B. Again rinse out syringe and microtip with sterile water and add 0. 025 ml serum C to rows 5 and 6 wells A-H, and row 11 wells A and B. (Row 9, 10, and 11 are serum controls). e. Tissue culture controls are the unused portion of the plate. f. Incubate virus and serum at room temperature for 30 minutes, then transfer reagents to cell cultures. SerumVirusSerum Controls A B C 123456789101112 ABC A |10-1 |No | |B |10-2 |Virus | |C |10-3 | | |D |10-4 Virus | | |E |10-5 | | |F |10-6 | | |G |10-7 | | |H |10-8 | | Beta Neutral ization Test Materials Needed: Flat-bottomed MT plate with lid 1 cc syringes MT tips Mt diluters sterile distilled water in beaker MEM diluent serum samples virus, 25-50 TCID50 bovine cell suspension Procedure: Use aseptic technique. a. Add 1 drop (0. 025 ml) diluent (MEM) to rows 1-8 wells A-H. b.Make 2-fold dilutions of serum through row H (final dilution 1:256), cleaning microdiluters in sterile distilled water between serums. c. Using the same syringe and microtip as in step a, fill with pretitrated (25-50 TCID50) IBR virus and add 0. 025 ml to rows 1-4 wells A-H, and to rows 7 and 8 wells A. d. Using rinsed microdiluters make 2-fold dilutions of the virus in rows 7 and 8 wells A-H. e. Incubate at room temperature for 30 minutes. f. Add 2 drops (0. 05 ml) of bovine cell suspension using a new 1 cc syringe and microtip to all wells of the test plus a few extra for tissue culture controls. Controls Serum ASerum BABVirus 123456789101112 A |1:2 | | | |B |1:4 |No | | |C |1:8 |Virus | | |D |1:16 | | | |E |1:32 | | | |F |1:64 | | | |G |1:128 | | | |H |1:256 | | | IV. CELL CULTURE Because viruses are obligate intracellular parasites, they cannot replicate in any cell-free medium, and thus require living cells from a suitable host within which to multiply.Animals such as mice and embryonating avian eggs may be used for the propagation of viruses, but for various reasons (time, cost, ease of handling, etc. ) the propagation of most viruses in a cultural medium of living cells is the method of choice today. More than half a century has elapsed since animal cells were first grown in vitro. In 1912 Carrel began growing bits of chick heart in drops of horse plasma. The cells at the edge of the explant divided and grew out of the plasma clot. The explants died within a few days, and Careel reasoned that their death was due to exhaustion of nutrients. He found that cells from a given explant could be maintained indefinitely if they were periodically subdivided and fed with a sterile aqueous extract of whole chick embryos.In the early 1950’s, Earle developed a technique for dissociating cells from a whole chick embryo from each other with trypsin. When this suspension of single cells was mixed with plasma and embryo extract and placed in a sterile glass container, the cells adhered to the glass and divided to form a primary culture. The primary culture contained a variety of cell types including macrophages, muscle fibers, etc. The cells grew to a monolayer, a thin sheet of cells (one layer in thickness) which covered the entire bottom surface of their culture vessel, and then stopped dividing. The cells could then be redispersed with trypsin and planted in new culture vessels containing fresh media.These secondary cultures contained fewer cell types than did the primary cell cultures, as many of the differentiated primary cells were out-competed and did not survive the transfer. Often, secondary cultures are composed entirely of spind le-shaped cells called fibroblasts because of their similarity to cultured connective tissue. Cells derived from kidneys and from certain carcinomas have a polygonal appearance in culture. Because of their tissue of origin, they and other cells with similar morphology are call epithelial. Cells may be grown in vitro in several ways. Organ cultures, if carefully handled, maintain their original architecture and functions for several days or sometimes weeks.Slices of organs (which are actually tissue cultures) consisting of respiratory epithelium have been used to study the histopathogenesis of infection by respiratory viruses that can only be grown outside of their natural host by using organ cultures. The term tissue culture was original applied to explants of tissue embedded in plasma. the term subsequently became associated with the culture of cells in general and is now obsolete in its original sense. Cell culture is the term most widely used today. It refers to tissue dissociate d into a suspension of single cells, which after being washed and counted, are diluted in growth medium and allowed to settle on to the flat bottom surface of a specially treated plastic or glass container.Most types of cells adhere quickly, and under optimum conditions they will undergo mitosis about once a day until the surface is covered with a confluent cell monolayer. There are three main types of cultured cells. The difference in these types lies in the number of times the cells can divide. 1. Primary cell cultures When cells are taken freshly from animals and placed in culture, the cultures consist of a wide variety of cell types, most of which are capable of very limited growth in vitro, usually fewer than ten divisions. These cells retain their diploid karyotype, the chromosome number and morphology of their in vivo tissues of origin. They also retain some of the differentiated characteristics which they possessed in vivo. Because of this, these cells support the replicatio n of a wide range viruses.Primary cultures derived from monkey kidney and mouse and chick embryos are commonly used for diagnostic purposes and laboratory experiments. 2. Diploid cell strains. Some primary cells can be passed through secondary and several subsequent subcultures while retaining their original characteristics. After 20-50 passages in vitro, these diploid cell strains usually undergo a crisis in which their growth rate slows and they eventually die out. Diploid strains of fibroblasts derived from human embryos are widely used in diagnostic virology and vaccine production. 3. Continuous cell lines. Certain cultured cells, notably mouse embryo fibroblasts and human carcinoma cells, are able to survive the growth crises and undergo indefinite propagation in vitro.After an initial slowing down, these continuous cell lines grow more rapidly than before, their karyotype becomes abnormal (aneuploid) and other poorly understood changes take place which make the cells immortal. The cells are now â€Å"dedifferentiated†, having lost the specialized morphology, and biochemical abilities they possessed as differentiated cells in vivo. Continuous cell lines such as KB and Hela, both derived from human others derived from mice (L929) and hamsters *BHK), are widely used in diagnostic and experimental virology. The development during World War II of antibiotics simplified long-term animal cell culture by minimizing the problems of bacterial and fungal contamination.Another important discovery was made by Eagle in the 1950’s when he determined the minimal nutritional requirements of cultured cells. He began by showing that Hela and Mouse L-cells would grow in a mixture of salts, amino acids, vitamins and cofactors, carbohydrates and horse serum. By eliminating one component at a time, he then determined which nutrients were essential for cell growth. His minimal essential medium (MEM) contains 13 amino acids (human tissue in vivo requires only 8), 8 vitamins and cofactors, glucose as any energy source and a physiological salt solution which is isotonic to the cell. The pH is maintained at 7. 2-7. 4 by NAHCO3 is equilibrium with CO2.The pH indicator phenol red is usually incorporated into the medium, which turns red-purple if the medium is alkaline, yellow if the medium is acidic, and remains red if the pH is suitable. Serum in concentrations of 1-10% must beaded to the medium to provide the cells with additional undefined factors, without which most cells will not grow. Most animal cells must be kept incubated at 37oC. If cells are grown in vessels open to the atmosphere, their incubator must be humidified and contain an increased CO2 concentration. Some nonvolatile phosphate or substituted sulfonic acid buffers (HEPES, TES) eliminate the requirement for incubators to be gassed with CO2. With the advent of cell culture, many animal viruses have been propagated in vitro, and hundreds of previously unknown viruses have been isol ated and identified.The discovery of the adenoviruses, echoviruses, and rhinoviruses, for example, is directly attributable to the use of cultured cells, as is the revolution in the diagnosis of viral diseases and the development of poliomyelitis, measles, and rubella vaccines. A. Culture of Primary Chick Embryo Fibroblasts (CEF) Materials 10-12 days old embryonated eggs Forceps and scissors Sterile petri dishes Sterile 250ml flask with magnetic bar Sterile 30 oz prescription bottles containing MEM & 5% lamb serum Sterile PBS Sterile 0. 5% trypsin (STV) Sterile 15ml centrifuge tubes containing 0. 5 ml serum Hemocytometers 1ml and 10ml pipets Sterile Dulbecco’s saline Procedure 1. Disinfect the surface of the egg over the air sac.With scissors or blunt end of forceps, break shell over air sac. Sterilize forceps by dipping in alcohol and flaming. Peel away shell over air sac, resterilize forceps and pull back shell membrane and chorioallantoic membrane to expose embryo. 2. Rest erilize forceps, grasp embryo loosely around neck, and remove from egg to sterile petri dish. 3. Using two forceps, or scissors plus forceps, decapitate and eviscerate embryo. Mince remainder of embryo to very small fragments. 4. Add about 10ml sterile Dulbecco’s saline to tissue fragments in petri dish, swirl to suspend fragments, and carefully pour into 250ml flask. With flask covered, continue swirling for 2-3 min. to wash tissue fragments.Tilt flask, allow fragments to settle, and gently decant saline. 5. Add 12ml sterile trypsin to fragments in flask, cover, and stir with magnetic bar for 15 min. Tilt flask, allow fragments to settle, and pour trypsin cell suspension into 15ml centrifuge tube containing 1ml serum. The serum contains a trypsin inhibitor which will prevent further damage to cell membranes but he enzyme (note: it is preferable to treat the tissue with multiple short applications of trypsin rather than a few long ones, in order to minimize enzymatic damage t o cell membranes. However, limitations of time require us to use the shorter method. ) 6. Add 12ml sterile trypsin to fragments and repeat step 5.At the end of this second treatment, size of tissue fragments would be greatly reduced and a large number of single cells should be suspended in trypsin. 7. Balance centrifuge tubes against one another and centrifuge at 1500 rpm for 10 min. Carefully decant off supernatant and resuspend pooled cell pellets in 1ml MEM. Make a 1:10 dilution of the cell suspension in MEM for counting in a hemocytometer. 8. In most hemocytometers each heavily etched square in 1mm on each side. The depth of the chamber is 0. 1mm. Count the cells in 0. 13 mm and calculate the number of cells in your original suspension. Dilute to give 8ml with 2-8 x 105 cells/ml in MEM, place in prescription bottle, replace cap tightly, and incubate on flat side at 37oC. 9. Be sure to examine cells periodically.Actively growing cells produce acidic metabolic by-products, and thu s the pH of the medium may need to be adjusted by the addition of a few drops of 7. 5% NAHCO3. If floating (dead) cells are present the medium may need to be changed. B. TRANSFER OF CELL CULTURES After cultured cells have formed a confluent monolayer on the surface of their culture vessel, they may be removed from the surface, diluted, and seeded into new vessels. If the initial culture was primary, the new cultures are called secondary, and are likely to consist of fewer cell types. Removal of cells from glass surfaces may be by either physical methods – scraping with a sterile rubber policeman – or chemical methods – proteolytic enzymes or chelating agents – or a combination of the two.After removal, cells are pipetted up and down and diluted appropriately in fresh secondary culturing, and after one becomes familiar with the growth characteristics of a certain cell types, counting can usually be dispensed with. We will transfer a cell line of bovine cel ls by use of a mixture of trypsin and EDTA (versene) in physiological saline (STV = saline, trypsin, versene): 1. Pour off the medium from a 3 oz. prescription bottle containing a confluent cell monolayer. 2. Wash the monolayer with 5-10 ml of physiological saline (Saline A) rinse well without shaking (shaking produces bubbles) and pour off. 3. Add 0. 5 ml STV to the bottle and incubate, with STV covering cells, at 37oC for 2-15 min.Observe periodically to determine when cells are loosened from glass (note: STV will contain a pH indicator and should have a pH of 7. 0-8. 0. Below pH 7. 0, trypsin is inactive. A pH above 8. 0 is damaging to cells. ) 4. When cells are seen to detach from glass upon shaking, add 6 ml fresh medium and suspend cells by pipetting up and down a few times. 5. Add 10ml more medium and mix to get even cell suspension. 6. Seed 1 ml cell suspension in to each of 8 culture tubes, stopper tightly, and incubate in rack which holds tubes at slight angle from horizon tal. Seed remaining 8 ml cell suspension into a new 3 oz. prescription bottle or a 25 cm2 plastic flask. C.PRESERVATION OFCULTURED CELLS BYFREEZING Viability of viruses and bacteria is preserved during freezing, but originally attempts to preserve animal cells by freezing resulted in cell death. This was first thought to be due to laceration of cell plasma membranes by ice crystals, but more recent evidence suggests the cause may be osmotic changes during freezing which give rise to irreversible changes in lipoprotein complexes in intracellular membranes. In any event, the answer to animal cell preservation has proved to be addition of glycerol, ethylene glycol, or dimethyl sulfoxide (DMSO) to the medium and slow freezing, ideally at a cooling rate of one centigrade degree per minute.Cells must be stored at 70oC or lower (ideally in liquid N2 at 196oC), and when they are recovered, thawing must be rapid. With careful technique, 50-80% of the cells of a healthy culture will survive f reezing. Procedure 1. Remove confluent cell monolayer from culture vessel by method described in cell transfer procedure. After centrifugation, resuspend cells in 1 ml medium containing 15% serum and 7. 5% DMSO and placed in small snap-top tube. 2. Immediately place tubes in an ice bath. They will then be transferred to a styrofoam container and refrigerated. After 20-30 min, when cells have dropped to 4o, they will be transferred to a 20o freezer for 20-30 min, then to the 70o freezer for storage.Alternatively, the tubes can be placed in cotton-or polystyrene-insulated containers and placed directly in the 70o freezer for slow cooling. If cells are to be stored in liquid N2, they must be placed in sealed ampoules. 3. To recover, cells, remove tubes from 70o and place directly in 37o water bath. When thawing is barely complete, add contents of tube to a 25 cm2 flask containing 15 ml MEM + 10% fetal calf serum. Culture medium will be changed for your approximately 4 hrs. later (after cells have attached) to reduce the toxicity of DMSO for cells at 37oC. D. Effect of Viral Infection on the Host Cell During the time that synthesis of viral components is occurring in the infected cell, the cell undergoes characteristic changes.These changes are usually observed in tissue culture where infection of cells is more easily synchronized and where the cells can be observed frequently during the course of infection. Morphological changes in cells caused by viral infection are called cytopathic effects (CPE): the responsible virus is said to be cytopathogenic. The degree of visible damage to cells caused by viral infection varies greatly. Some viruses cause very little or no CPE. Their presence can be detected only by hemadsorption (already discussed) or interference, in which infected cell cultures showing no CPE inhibit the replication of another virus subsequently introduced into the cultures.On the other hand, some viruses cause a complete and rapid destruction of the cell monolayer after infection. The histological appearance of the CPE caused by some of these cytocidal viruses may be sufficiently characteristic to allow provisional identification of the virus. Some CPE can be readily observed in unfixed, unstained cells, under low power of the light microscope, with the condenser down and the iris diaphragm partly closed to obtain the contrast needed for viewing translucent cells. Several types of CPE are distinguishable in living cultures, but fixation and staining of the cells is necessary to see such manifestations of viral infection as inclusion bodies and syncytia.Recognizing CPE and using it as a diagnostic tool requires much experience in examining both stained and unstained cultures of many cell types. Listed below are several general types of CPE. Keep in mind that a given virus may not conform to the norm for its family, or it may produce different CPE in different host cell types. The best knowledge of viral CPE comes from experience . 1. Total destruction of the cell monolayer is the most severe form of CPE. All cells in the monolayer rapidly shrink and become dense (Pyknosis) and detach from the glass within 72 hours. This CPE is typical of most enteroviruses. 2. Sub-total destruction consists of detachment (death) of some but not all of the cells in the monolayer.The alpha-togaviruses, some picorna viruses, and some of the paramyxoviruses may cause this type of CPE. 3. Focal degeneration is characteristic of the herpesviruses and poxviruses. Instead of causing a generalized destruction of the cell monolayer, these viruses produce localized areas (foci) of infection. The focal nature of these lesions is due to direct cell-to-cell transfer of virus rather than diffusion through the extra-cellular medium. Cells initially become enlarged, rounded, refractile (more easily seen), and eventually detach from the glass, leaving cleared areas surrounded by rounded up cells as the infection spreads concentrically. Stran ding of the cytoplasm is usually pronounced and cell fusion may be evident. 4.Swelling and clumping of cells before detachment is typical of adenoviruses. Infected cells greatly enlarge and clump together in â€Å"grape-like† clusters. 5. Foamy degeneration (vocuolization) is due to the production of large and/or numerous cytoplasmic vacuole. Several virus families including certain retroviruses, paramyxoviruses, and togaviruses may cause vocuolization. 6. Cell fusion (syncytium or polykaryon formation) involves the fusion of the plasma membranes of 4 or more cells to produce one enlarged cell with 4 or more nuclei. Polykaryon formation may be the only detectable CPE of some paramyxoviruses; herpesviruses may also produce syncytia. 7. Inclusion bodies are areas of altered staining in cells.Depending on the causative virus, these inclusions may be single or multiple, large or small, round or irregularly shaped, intranuclear or intracytoplasmic, eosinophilic (pink staining) or basophilic (blue-purple staining). In most cases they represent areas of the cell where viral protein or nucleic acid is being synthesized or where virions are being assembled, but in some cases no virus is present and the inclusion bodies represent areas of viral scarring. V. BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF VIRUSES There are many biochemical and biophysical tests which can be used for classification of viruses. We will perform four of these test using â€Å"unknown† viruses: viral sensitivity to lipid solvents, determination of virus size, determination of virus nucleic acid type, and viral sensitivity pH and heat.The chart on p. 127 of your lab book may help in the identification of your virus. A. Viral Sensitivity to Lipid Solvents. The lipid sensitivity test is one of the most basic tests for characterization of viruses. There is a correlation between the presence of an envelope and the susceptibility of viruses to lipid solvents such as ether, chloroform, and detergents. Enveloped viruses require their lipid membrane for infectivity; because the test measures destruction of viral infectivity vs. untreated viral controls, it is an indirect test. All lipid coated viruses are sensitive to chloroform, whereas all but a few poxviruses are sensitive to ether.This is because the lipid components of the poxviruses are much diffe

El Paraiso Hotel Resort Cebu and Its Health Benefits

Profile of the Company El Paraiso Hotel Resort Cebu is a five star hotel which offers comfortable place and quality services to make the stay of the guests a pleasurable one. It is located in Lapu-Lapu City, which is composed of several small islands and connected to mainland Cebu by two bridges, the resort is just 30 minutes away from the international airport. Being one of the most luxurious hotels in the Philippines, making the vacation of guests a memorable one is one of the main objectives of the company. Vision of the CompanyEl Paraiso Hotel and Restaurant as the number one provider of quality hotel and food services within the islands of the Philippines. Mission of the Company El Paraiso Hotel and Restaurant aims to provide quality hotel and food services through regular and continuous employment, trainings, seminars and supervisions of highly qualified, efficient and good-natured employees and through the use of state-of-the-art facilities and convenient hotel rooms, kitchens , lobbies, restaurants and other hotel places. Hotel Facilities * Infinity pool * Fitness center * Wellness center * Cinema * Souvenir shop * Clinic * 3 restaurants and 1 bar Lobby lounge * 186 rooms PROJECT HEAD * Elaine Michellica Lopez (Human Resource Manager) * Lovely Cabacoy (Assistant HR Manager) * Krystel Urbina (Assistant HR Manager) * Sharmaine Orense (Project Development Supervisor) * Christchen Del Valle (Research and Extension Coordinator) * Joan Tolentino (Assistant R&E Coordinator) * Marc Kenneth Quitalig (Assistant R&E Coordinator) BUDGET The company has allotted six hundred twenty five thousand pesos for this project. This includes four hundred seventy five thousand pesos for the wellness project and two hundred thousand pesos for the health program.Monthly expenses will be incurred for this project that is why the company will set other funds for these expenses. RESOURCE NEEDED Financial Resources The financial resources will be coming from the fund set aside solely for the corporate social responsibility programs of the company. In view of the fact that this company has been operating for many years now, it is expected that it has allotted funds for this kind of projects because in the long run, this project will benefit not just the employees but the company as well. Human ResourcesThe top management should be the first one to cooperate in this project. Their approval will be the key factor on the possibility of this project to put into action. The project coordinators should do their best to put in practice the strategies needed for the implementation of this program. They must thoroughly follow the set guidelines on how they will perform their individual duties and responsibilities. The employees benefited should also cooperate in this project. They must exert their full understanding and appreciation on the project provided for them.While the company is exerting effort on how they can improve or at least maintain their healthy lifestyle, they must be disciplined even in their respective homes. Discipline is a must in this project. ORGANIZATION TIE-UPS Different departments and agencies are going to be involved in this project. These are the Department of Tourism, Department of Health, Department of Labor and Employment, Bureau of Food and Drugs, UNILAB and Mactan Doctors’ Hospital. These departments and agencies have their own part in the realization of this program. Department of Tourism is concerned about the tourists that are going to be the customers of the hotel.Department of Health is concerned about the health of the employees providing the services for customers while Department of Labor and Employment is concerned about the overall condition of the employees in the company. The Bureau of Food and Drugs will serve as the guide of the company in the distribution of vitamins and medicines to the employees. UNILAB will be the reliable supplier of the company of the vitamins and medicine to be distributed and the Mactan Doctors’ Hospital is going to be partner of the company in medical examinations that will be done to every employee.PROGRAM PLAN I. Title of the Project This project was entitled as â€Å"Wealthness at El† because it focuses on the health and wellness of all the employees of the company. The company is concern with the health and wellness of its employees because the company believes that they provide great contributions to the success of the company just like other stakeholders. II. Location The management of El Paraiso decided to propose this Corporate Social Responsibility Program in Mactan, Cebu since Cebu is one of the main tourist spots here in the Philippines.Having an international airport in Mactan, Cebu is a great factor to the operations of the company. Employees need to face different types of people from different cultures and status in life. III. Duration As long as the company is operating and obtaining enough profit to sustain the projec t, the management believes that this program will continue to exist. If the management will approve of this project, it is expected to be established on the first quarter of the following year. Since the first project is about forming a company fitness and wellness center, it is expected to operate as long as the company is in service.With regards to the next project which is about the medical assistance and support for employees, the proponents foresees that as long as the company is generating large profit from its operations, the project will continue. According to the top management of the company, it is expected that for the next 50 years, the company will continue to operate. IV. Type of the Program The proposed project is a type of Health and Wellness Program for all the employees of El Paraiso. This is the type of program we have proposed because being with people of all raises requires enough strength and confident aura.This will only be possible if the employees facing the m are very much healthy and well disciplined in terms of physical health. Less absences and tardiness and better performance will be the benefit that the company will get from this project. In the short run, the benefit against cost cannot be easily achieved but in the long run, the company believes that the initial cost of this project will be insignificant if it will be measure against the benefit that the company and the employees will get from its employees. V. Department Involved * Department of Health the executive department of the Philippine Government responsible for ensuring access to basic public health services to all Filipinos through the provision of quality health care and the regulation of providers of health goods and services. Since once of the project proposed involves that health of the employees, it is only right to involve the Department of Health in this project. * Department of Tourism * The executive department of the Philippine government responsible for th e regulation of the Philippine tourism industry and the promotion of the Philippines as a tourist destination.The nature of the company is a hotel and resort. Different tourists around the world are going to be the customers of the company. Department of Tourism should be involved in this project because their main concerns are the tourists and tourist spots in the Philippines. * Department of Labor and Employment * the executive department of the Philippine Government mandated to formulate policies, implement programs and services, and serve as the policy-coordinating arm of the Executive Branch in the field of labor and employment.The main beneficiaries of these projects are the employees of the company. Since it is a private corporation it is the Department of Labor and Employment who has the concerned over the employees of the company. VI. Project Leader and Coordinators Project leader: Elaine Michellica Lopez (HR Manager) Coordinators:Lovely Cabacoy (Asst. HR Manager) Krystel U rbina (Asst. HR Manager) Sharmaine Orense (PD Supervisor) Christchen Del Valle (R & E Coordinator) Joan Tolentino (Asst. R&E Coordinator) Marc Kenneth Quitalig (Asst. R&E Coordinator) VII. Cooperating Agencies * Mactan Doctors’ Hospital a 151 bed tertiary infirmary fully equipped with the latest cutting edge technology for your optimum diagnostic and therapeutic treatment under the care of their elite medical and surgical specialists. In case that the clinic of our company cannot support the health requirements needed by our employees, they can visit this hospital for further examinations. They can avail discounts if they present the health cards distributed by the company. * UNILAB * the Philippines‘ largest and leading pharmaceutical and healthcare company with over 60 years experience of giving the  best value in healthcare to Filipinos.The vitamins that the company needs in this project will be directly coming from UNILAB. Since there is going to be a contact abou t the supply of vitamins, discounts will be expected from the transactions. * Bureau of Food and Drugs (BFAD) * a regulatory agency that ensures the safety, efficacy, purity and quality of processed foods, drugs, diagnostic reagents, medical devices, cosmetics and household hazardous substances through state-of-the art technology, as well as the scientific soundness and truthfulness of product information, for the protection of public health. VIII. BeneficiariesAll the employees of El Paraiso Hotel and Restaurant Cebu Branch are going to be benefited from this program. This program is proposed for the well being of the employees working for the company because they are the assets of the company. For the company, the employees are one of the major reasons why the company is continuously operating and earning profit. It is only right that they get benefits from the company concerning their health and wellness. IX. Total Cost of the Project The estimated initial cost of the first proje ct for the wellness of the employees cost four hundred fifty thousand pesos (Php 450000. 0). For the next project which is for the health of the employees has an initial cost of around one hundred seventy five thousand pesos (Php 175000. 00). The total initial cost for the project proposed is six hundred twenty five thousand pesos (Php 625000. 00) The monthly budget for the health assistance provided for employees costs two hundred twenty thousand pesos (Php 220000. 00) X. Rationale of the Project Our company believes that our employees are the asset of the organization. We value them in the best possible way because they are of great contribution to the success of the company.To show our concern to them, we decided to have a program that will address their health issues. It is very important and an advantage of the company to have healthy people inside the organization. This program ensures that their health status is regularly monitored. The company also implements this program no t just to ensure health awareness but also to motivate the employees that will result to increasing productivity and reduces cases of absenteeism and tardiness. XI. Objectives General Objectives: * To maintain a healthy workforce who would therefore provide quality ervice to customers * To provide continuous benefits to every employee of the company * To improve the well being of every employee * To prevent and manage any form of health difficulties or disease Specific Objectives: * To provide fitness centers that would be accessible to every employee * To provide monthly check-up for every employee and supply of vitamins monthly * To give health cards to every employee wherein they can avail discounts on hospitals and medicine. XII. Description of the Project Strategies and MethodsIn order to aid the management in the execution of the said project, the following strategies were formulated: 1. Information of this project would be properly disseminated so that every employee would be informed of the benefits that they could get from the said project. 2. The company would have a partnership or coordinate with a trusted hospital to aid it on the monthly check up of the employees. 3. It would also coordinate with a drug manufacturer and buy drugs directly to it to ensure quality of the drugs and it is also a cost saving move. . The management would also keep track of the health records of the employees by providing each employee with a medical journal on which the results of their monthly check up would be written. 5. The management would establish a schedule for the monthly check up of the employees. For instance, a group of employees would be scheduled for their check up for a certain week and so forth. This is to ensure that the monthly check up would be well organized and would not be burdensome on the part of the physicians. 6.For a more effective distribution of vitamins, the employees would be given their monthly supply of vitamins during their scheduled ch eck up. 7. The management would make it clear that the fitness center is accessible to every employee and they could use it anytime as long as it is not during working hours. XIII. Financial Plans WELLNESS PROJECT Total Initial Cost of the Project| Total Cost of Gym Equipments| 269000| Installation Costs| 50000| Renovation (lighting and ventilation) of the function hall| 100000| Total initial cost of wellness program| 419000| GYM EQUIPMENTS| PRICE| QUANTITY| TOTAL COST|Treadmill| 15000| 5| 75000| Exercise bike| 10000| 5| 50000| Upper body trainer| 12000| 5| 60000| Dumb bells| 1000/pair| 20| 20000| Punching Bags| 3000| 3| 9000| Gloves and Mitts| 1000| 10| 10000| Boxing balls and swivels| 3000| 3| 9000| Table tennis table| 8000| 2| 16000| Table tennis rackets| 2000/pair| 10| 20000| Total Cost| | | 269000| HEALTH PROJECT Total Initial Cost of the Project| Renovation (lighting and ventilation) of the clinic room| 50000| Total cost of medical equipments| 78600| Cost of health cards and j ournals to be distributed among employees| 10000| Installation of medical equipments| 30000|Total Initial Costs| 168600| MEDICAL EQUIPMENTS| TOTAL COST| Wheelchairs (3 pcs)| 21000 | Hospital bed (2 pcs)| 30000| Blood pressure monitor (3 pcs)| 6600| Nebulizer (2pcs)| 9000| Weighing Scale| 4000| Gauze, tape and bandages| 3000| Latex and vinyl gloves| 3000| Thermometers| 1000| Disinfectants| 1000| Total Cost| 78600| Financial budget for every month Total Cost of Vitamins to be distributed monthly| 100000| Fund for the hospitalization of employees per month| 50000| Monthly salary of the physician and assistant nurses| 70000| Total| 220000|XIV. Functional Relationships with the Collaborating Agencies El Paraiso is proud to implement the â€Å"Wealthness at El† for ensuring the health and wellness of every employee. In line with this, our company had its partnership with different agencies that would be a great contributor for the implementation of the program. These agencies are t he Mactan Doctors’ Hospital, UNILAB, and Bureau of Food Drugs. First, we conducted our board meeting together with the head from each respective agency. Every matter concerning this program had been discussed.Then we had the contract signing thereafter. Our agreement is that they would continue supporting our program as long as it continues its operations. The monthly check-up of the employees would not be possible without the staff coming from the hospital. This includes board certified physicians in internal medicine and occupational medicine (Preventive Medicine), doctors and registered nurses who provide a variety of direct health care services and health promotion activities for the company. The vitamins and other medicines needed would be coming from the drug manufacturer.The company decided to have its partnership directly from the manufacturer to obtain quality products yet affordable, so that this program would not be that costly. XV. Monitoring and Evaluation Mechan ics In order to monitor and evaluate the performance of the said project, the management had established the following mechanics: 1. The management would provide evaluation sheets which the employees would answer with the following questions: * Are you aware of Wealthness at El? * Are you able to benefit from the said project? In what ways did it become beneficial to you? Do you have any complaints regarding the procedure and other matters about the project? What are they? * Do you have any suggestions that the management could do to respond on these complaints? * What can you recommend to the management to further improve this project? 2. The head physician would periodically report to the management and discuss the matters of the project particularly their health status. XVI. Plans for Ensuring the Sustainability of the Project In order for a business to be successful in the long run, they need to contribute to the welfare of the community, conservation of the environment and moti vation of their employees.This is what we called the Corporate Social Responsibility (CSR). Our company was concerned with our employees because they are one of the contributors why our business continues to operate. That’s why we have proposed a project for them. We want them to obtain some other benefits on our company while contributing to the success of our business. For us to ensure the sustainability of our project, we have made several plans. Those are the ff: * During the mere operation, we will provide a separate fund or will restrict our fund for the sustainability of this project. We are going to sustain this through obtaining a contingency fund. * We will be having a deal with our collaborative agencies so that they will support our needs to sustain it. * Since our company has been operating for almost decades, we are well-known in the country. We will be having sponsors so that we didn’t need to worry that much. * In case of loss, our main branch located i n Manila is the one to help us recover so that we will continue operating. PROGRAM EVALUATION Based on the survey conducted by the company and based on the medical journals of the employees, the results showed improvement of their respective health status.This project has been implemented on every employee of the company. Schedules of check-ups were implemented so that it was certain that every employee has undergone medical examinations. The vitamins that have been distributed to the employees are effective in improving their energy towards work which resulted to better performance in terms of providing quality service to customers. In terms of the wellness program, the healthy lifestyle of the employees has been maintained. Sickness has been avoided and their confidence towards their looks and appearance has increased. t was found out that certain improvements will be needed for the betterment of the techniques and strategies needed to make this project successful. More systematic strategies should be needed for the distribution of vitamins and schedules of check-ups so that delays can be avoided. After several years of execution of this project, the company had been benefited because it is able to maintain a healthy workforce that would provide its customers quality service. The employees of the company are able to receive the benefits that these particular project aims to provide to them.Company Benefits from CSR Program Through the â€Å"Wealthness at El†, the company is able to derived the following benefits: 1) The company is able to maintain a healthy workforce who will provide quality service to the customers. 2) Its employees are more motivated because they feel that the company value them. 3) Motivated employees will result to increase in productivity and reduce cases of absenteeism and tardiness. 4) Therefore, a healthy and motivated workforce will result to the company’s improved reputation which enables them to attain higher income.

The Works of Kate Chopin essays

The Works of Kate Chopin essays Women writers in the late 19th century struggled with their voices, their characters, and their writing, just as all writers do. However, many also had to struggle to be taken seriously by predominately male publishers, and such was the case with Kate Chopin. Throughout her career, "Chopin wrote three novels, eighty-five short stories, twelve essays, twenty-five poems, and one play, although she never earned enough money to support herself from her writing" (Nelson, 1995 p. 234). The key here is "support herself." It is still quite difficult to support oneself from fiction writing, and Chopin encountered this throughout her career. Quite simply, short stories sold more quickly than novels, and took less time to write, so Chopin could create more marketable works in a shorter amount of time, and see the monetary results much sooner than she would from a novel. As one writer noted, "The market for conversion and happily-ever-after stories for Christmas and Easter was immense. It was also one of the best sources of income and recognition for professional writers" (Toth, 1999 p. 125). Indeed, Chopin consistently made more money from her short stories than her novels. She received only $102 as royalties for "The Awakening," but at the same time was earning as much as $50 for only one short story (Toth, 1999 p. 229). Historian Toth continues, "Magazine editors in the 1890s, all of them white, liked stories of unequal relationships between blacks and whites in which, for instance, the black character sacrifices for his or her 'white folks'" (Toth, 1999 p. 169). Fiction was exceedingly popular in women's magazines of the day, and as more magazines sought to capitalize on the women's market, more fiction was needed to fill their pages, and so the opportunities for short story writers were quite good. Good Housekeeping was quite consistent with other women's magazines of the ...

Lajom, John Froilan C. Oral Communication Essays - Cultural Studies

Lajom, John Froilan C. Oral Communication Essays - Cultural Studies Lajom, John Froilan C. Oral Communication S11-08 How is intercultural communication exhibited in the film? Cite two scenes to support your answer. As we know intercultural communication is communication between at least two people from different culture. From the time Anna somewhat unfortunately landed to Wales and started to be with the Irish people, intercultural communication was display. One scene that exhibits this kind of communication is when she talked to Declan and the elders about her planned proposal to his boyfriend and how and when Declan and Anna will be able to go to Dublin. The elders are trying to say the day that is not advisable to travel and Anna, being new to the town just pretend to listen which is not good to intercultural communication. Another scene is the first parts of the journey of Declan and Anna to Dublin when they find that the herd of cows has blocked the road, Declan instantly sits down and wait while Anna attempted to get the cows to move. Nonverbally intercultural communication was not exhibited very well because they don't connect a channel to communicate; Declan is carefree while Anna is al ways in a hurry. Also Declan switch to his own language while talking to Anna by saying Bob which means money this is not right for intercultural communication. Describe how Anna communicated with the Irish people. Anna is not really an effective communicator with the Irish people. It seems like she doesn't know how to interact intercultural because she interfere what Irish people are and their cultural dimensions. It's not that she is not polite when approaching them, but she gave them not-so-good impression. She is bland and sound like always in a hurry and unwilling to defer gratification. She always follows her "strict time schedule". That is only at the first parts of the film because as the story goes, she started to go along with the polychromic environment of the Irish people. Maybe she's just not that used to the carefree spirit of Irish people, especially Declan, making a difference in the message she is trying to convey. Identify the effective communication skills (i.e.. eye contact visible mouth, body language, silence, checking for understanding, smiling face, summarizing what has been said, encouragement to continue, some questions) applied by a character in the movie. Justify your answer. Although not one of the main characters the wife of the train station employee shows effective communication skills to Anna and Declan. When talking she speaks while looking at the person's eyes and speaks clearly. She smiles frequently (and appropriately) giving the two visitors a warm feeling and welcome. She also listens whenever any of the two speaking like for example, when Declan was taking a shower. She also shows her authenticity by being real to the two like when she shows the two how to kiss their loved ones. Also, she asked questions to continue the conversation and make it more pleasing. All in all she communicates with them despite difference in culture and perspective.